Structure of human ADP-ribosyl-acceptor hydrolase 3 bound to ADP-ribose reveals a conformational switch that enables specific substrate recognition

Abstract

ADP-ribosyl-acceptor hydrolase 3 (ARH3) plays important roles in regulation of poly(ADP-ribosyl)ation, a reversible post-translational modification, and in maintenance of genomic integrity. ARH3 degrades poly(ADP-ribose) to protect cells from poly(ADP-ribose)-dependent cell death, reverses serine mono(ADP-ribosyl)ation, and hydrolyzes O-acetyl-ADP-ribose, a product of Sirtuin-catalyzed histone deacetylation. ARH3 preferentially hydrolyzes O-linkages attached to the anomeric C1″ of ADP-ribose; however, how ARH3 specifically recognizes and cleaves structurally diverse substrates remains unknown. Here, structures of full-length human ARH3 bound to ADP-ribose and Mg²⁺, coupled with computational modeling, reveal a dramatic conformational switch from closed to open states that enables specific substrate recognition. The glutamate flap, which blocks substrate entrance to Mg²⁺ in the unliganded closed state, is ejected from the active site when substrate is bound. This closed-to-open transition significantly widens the substrate-binding channel and precisely positions the scissile 1″-O-linkage for cleavage while securing tightly 2″- and 3″-hydroxyls of ADP-ribose. Our collective data uncover an unprecedented structural plasticity of ARH3 that supports its specificity for the 1″-O-linkage in substrates and Mg²⁺-dependent catalysis.

Keywords: ADP-ribosylation; ARH3; PARP1; conformational change; hydrolase; structural biology; substrate specificity.

Figure 1.

Structure of full-length human ARH3 bound to ADP-ribose and Mg²⁺. a, structures of ARH3 substrates: a linear unbranched poly(ADP-ribose) (left), O-acetyl-ADP-ribose (middle), and α-ADP-ribosylserine (right). ARH3 cleaves the 1″-O-linkage in substrates. The exoglycohydrolase activity of ARH3 cleaves the α(1″-2′) O-glycosidic bond between n and n − 1 ADP-ribose, releasing ADP-ribose as a product. b, left, Mg²⁺ enhances the ADP-ribosyl-acceptor hydrolase activity of ARH3. The ARH3-mediated hydrolysis of PAR on PARylated PARP1C was monitored in the presence and absence of Mg²⁺ using a gel-based assay. PARG has stronger PAR turnover activity than ARH3 and was used as a positive control. Right, metal preference of ARH3. ARH3 prefers Mg²⁺ for catalysis followed by Mn²⁺ and Ca²⁺. c, difference electron density maps (F_o − F_c) for ADPR and Mg²⁺ ions contoured at 3.0 σ (blue, ADPR; orange, Mg²⁺). d, structure of the ARH3–ADPR–Mg²⁺ complex revealing two unique and flexible structural elements, adenine cap (green) and glutamate flap (Glu⁴¹-flap) (red), that undergo conformational changes and strongly contribute to specific substrate recognition. The 1″-OH of ADPR (blue circled), corresponding to the scissile 1″-O-linkage in substrates, is exposed to solvent, consistent with ARH3 specificity for 1″-O-linkage for cleavage. A putative binding site for the leaving group is highlighted with a black ellipse. The N and C termini of ARH3 are indicated by N and C, respectively.

Figure 2.

Structure-based alignment of ARH3 and DraG with structural elements and residues that are important for function. The Glu⁴¹ residue of the Glu⁴¹-flap of ARH3 that undergoes a large conformational change upon ADPR binding is indicated by a red box. A part of L2 of DraG (L2 wall) completely blocks the conformational change of α1 and restricts its activity to cleavage of mono(ADP-ribosyl)ated substrates. The end of α1 in ARH3, which exists as 3₁₀-helix in the unliganded form and undergoes 3₁₀-to-α transition upon ADPR binding (Fig. 4a), is indicated by a blue bar. Two aromatic residues in DraG that stabilize the L2 wall are indicated by a red triangle. Asp⁹⁷ in DraG that is essential for the cleavage of MARylated arginine is indicated by a blue triangle.

Figure 3.

ARH3 specifically exposes the scissile 1″-O-linkage in substrates for cleavage. a, structural superposition of ADPR-bound forms of ARH3 (wheat) and DraG (gray) reveals a distinctive ADPR-binding mode in ARH3. The adenine cap of ARH3 (green) grasps the adenine ring and is essential for ARH3 activities. Hydrogen bonds contributed by the main-chain atoms of the adenine cap to N6 and N7 of the adenine ring impart specificity. b, diagram showing interactions between ADPR and ARH3. c, close-up of the binuclear Mg²⁺ catalytic center and ADPR binding in ARH3. A matrix of Mg²⁺-mediated coordination and hydrogen-bonding interactions secure ADPR in the active site, which is consistent with Mg²⁺-dependent catalysis by ARH3. The 1″-OH of ADPR (blue circled), corresponding to the scissile 1″-O-linkage, is specifically exposed to solvent, strongly supporting ARH3 specificity for the 1″-O-linkage as a site for cleavage. d, close-up of the ARH3–di-ADPR–Mg²⁺ complex model. The energy-minimized computational model for the ARH3–di-ADPR–Mg²⁺ complex was generated using the ARH3–ADPR–Mg²⁺ complex as a starting model (see “Experimental procedures”). In this model, 3′-OH of the n − 1 ADPR leaving group is directly coordinated by Mg^B, replacing the W1 water ligand, and Mg^B tightly secures both n and n − 1 ADPR units for efficient cleavage to occur.

Figure 4.

A conformational change of ARH3 enables specific substrate recognition and cleavage. a, structural comparison of the unliganded (gray/black) and ADPR-bound forms (orange/red) of ARH3 reveals a conformational switch in the Glu⁴¹-flap. The end of α1 undergoes a 3₁₀-to-α transition upon ADPR binding, which results in displacement of Glu⁴¹ away from Mg^B and significantly widens the leaving group–binding site of ARH3. b and c, ARH3 switches conformations to enable specific poly(ADP-ribose) recognition. The structure of the ARH3–di-ADPR–Mg²⁺ complex model is superimposed onto those of the ARH3–ADPR–Mg²⁺ complex and the apo-ARH3^ΔN16, and di-ADPR is shown on the surfaces of ARH3. Comparison of solvent-accessible surfaces reveals an open platform that can accommodate n − 1 ADPR in the ARH3–ADPR–Mg²⁺ complex (b), whereas n − 1 ADPR binding is completely obstructed by the Glu⁴¹-flap in the unliganded ARH3 (c).

Figure 5.

Asp314 is essential for the formation of the binuclear metal center. Structural comparison of the WT and catalytically inactive ARH3-D314E mutant reveals a side-chain flipping of Glu³¹⁴, which leads to the loss of Mg^B. This suggests that Asp³¹⁴ is essential for the formation of the binuclear metal center in ARH3 and that Mg^B is required for catalysis.

Figure 6.

ARH3 switches between closed and open conformations to specifically recognize and cleave substrates. ARH3 switches between a self-inhibited and an incision-competent conformation for specific substrate recognition and cleavage. In the absence of PAR substrates, the Glu⁴¹-flap caps Mg^B, constituting a closed, self-inhibited enzyme state. Substrate binding induces a conformational switch in the Glu⁴¹-flap, allowing substrate entrance to the active site. This active-site rearrangement in ARH3 specifically exposes the scissile 1″-O-linkage to the catalytic Asp³¹⁴ for cleavage, explaining ARH3 specificity for the 1″-O-linkage in structurally diverse substrates.